Efficient production of 1,2,4-butanetriol from corn cob hydrolysate by metabolically engineered Escherichia coli

Corn cob is a major waste mass-produced in corn agriculture. Corn cob hydrolysate containing xylose, arabinose, and glucose is the hydrolysis product of corn cob. Herein, a recombinant Escherichia coli strain BT-10 was constructed to transform corn cob hydrolysate into 1,2,4-butanetriol, a platform substance with diversified applications. To eliminate catabolite repression and enhance NADPH supply for alcohol dehydrogenase YqhD catalyzed 1,2,4-butanetriol generation, ptsG encoding glucose transporter EIICBGlc and pgi encoding phosphoglucose isomerase were deleted. With four heterologous enzymes including xylose dehydrogenase, xylonolactonase, xylonate dehydratase, α-ketoacid decarboxylase and endogenous YqhD, E. coli BT-10 can produce 36.63 g/L 1,2,4-butanetriol with a productivity of 1.14 g/[L·h] using xylose as substrate. When corn cob hydrolysate was used as the substrate, 43.4 g/L 1,2,4-butanetriol was generated with a productivity of 1.09 g/[L·h] and a yield of 0.9 mol/mol. With its desirable characteristics, E. coli BT-10 is a promising strain for commercial 1,2,4-butanetriol production. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-024-02317-0.


Introduction
Corn is one of the most important sources of starch in the world.Corn cob is inevitably and massively generated as a by-product of the corn agriculture.Nowadays, corn cob is either burnt as fuel or treated as a waste causing environmental pollution [1,2].Corn cob can be hydrolyzed into corn cob hydrolysate (CCH) with xylose, arabinose and glucose [1,2].Numerous efforts have been made to obtain efficient routes for resource utilization of CCH [3][4][5].Many value-added chemicals including xylitol, [6] xylonate, [7] and ethanol [8] can be produced from CCH by metabolic engineered microorganisms.

Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1.E. coli strains were generally cultivated in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 180 rpm and 37 °C.The pTKRED and pCP20 were used for the gene knock-out or knock-in of E. coli W3110 (DE3).Kanamycin, chloramphenicol and spectinomycin were added at a concentration of 50, 40, and 50 μg/mL when necessary.

DNA manipulation in E. coli W3110 (DE3)
The primers used are listed in Table S1.Vector isolation, restriction enzyme digestion, agarose gel electrophoresis, and other DNA manipulations were carried out using standard protocols as in our previous study [7].Plasmid was transformed into chemically competent cells by heat shock, and transformants were isolated by plating on antibiotic LB-agar plates.The genes xylB, xylC, xylD and kdcA were synthesized by Tongyong Biosystem Co., Ltd.(Chuzhou, Anhui, China) in our previous study [7,27].The Red recombination technology was applied for knockout of genes pgi and ptsG, and knock-in of genes xylBC from Caulobacter crescentus in E. coli W3110 (DE3) [26].The fragment ΔptsG used for knockout of ptsG was obtained by directly amplify the fragment with primers ΔptsG-F1/ΔptsG-R1 from E. coli  S1) were used to clone up homologous arm of xylA, the genes of xylBC, kanamycin resistance gene, and down homologous arm of xylA.These four fragments were recombined to form fragment ΔxylA::xylBC.The fragments Δpgi for knockout of pgi, ΔptsG::P T7 -xylBC and ΔmgsA::P T7 -xylBC for knock-in of genes xylBC at the position of genes ptsG and mgsA were obtained through similar process.The fragments for knockout or knock-in of different genes were transformed into E. coli cells containing pTKRED plasmid by electrotransformation.Positive transformants were selected by relevant antibiotics and confirmed by PCR and subsequent DNA sequencing.Then, the plasmid pCP20 was transformed to eliminate the kanamycin resistance gene kan from chromosome.The two temperature-sensitive plasmids, pTKRED and pCP20, were removed by culture at 42 °C overnight.

Batch fermentations and fed-batch fermentation
Batch fermentation was carried out in 300 mL shake flasks containing 50 mL of LB medium with 10 g/L xylose and 2 g/L lactose at 180 rpm and 37 °C for 24 h.Xylose at the concentration of 10 g/L was added in the medium for fed-batch fermentation in shake flask when necessary.Fed-batch fermentation was also conducted in 1.0-L bioreactor (Infors AG, Bottmingen, Switzerland) with an operating volume of 0.8 L or 7.5-L (B.Braun Biotech International GmbH, Germany) bioreactor with an operating volume of 5 L, respectively.The seed was inoculated directly into a 35 mL test tube containing 5 mL of the LB medium and then was cultivated at 180 rpm in a rotary shaker at 37 °C overnight.The seed culture was prepared in a 500 mL shake-flask containing 100 mL LB medium, and then seed culture was inoculated (10%, v/v) into the fermentation medium.The LB fermentation medium contained 10 g/L lactose, 10 g/L glucose and 30 g/L xylose.Alternatively, the detoxified CCH (25%, v/v) and whey powder (12.99 g/L) were added into the broth to make the xylose concentration at about 30 g/L and lactose concentration at about 10 g/L.Fermentation was performed at 30 °C with an aeration rate of 1.5 vvm and an agitation of 400 rpm.Samples were withdrawn periodically to determine the cell density, concentrations of glucose, xylose, arabinose, lactose, 1,2,4-BT and by-products.When xylose concentration was lower than 10 g/L, xylose or CCH was added to make xylose concentration to 30 g/L.The pH was maintained at 7.0 by automatic addition of 10 M NaOH.

Construction of 1,2,4-butanetriol biosynthesis pathway in E. coli
Biosynthesis of 1,2,4-BT from xylose and arabinose involves five identical enzymic steps including dehydrogenation, hydrolysis, dehydration, decarboxylation and reduction (Fig. 1).In this study, the synthesis pathway for production of 1,2,4-BT from xylose, the most abundant carbohydrate in corn cob, was firstly constructed in E. coli W3110 (DE3) (referred to herein as 0K).E. coli 1K, 3K, 4K and 4KI were four derivative strains of E. coli 0K constructed previously [27].E. coli preferentially utilizes glucose in medium containing glucose and other utilizable sugars like xylose and lactose [24].Knocked out the ptsG encoding the glucose-specific transporter EIICB Glc can eliminate carbon catabolite repression of glucose.Thus, E. coli 5KI was constructed by deleting ptsG in E. coli 4KI and then the plasmid pETP tac -xylBC was transformed, resulting in strain E. coli BT-6.Glucose can also be transported by galactose symporter after deletion of ptsG gene but the glucose utilization rate of the constructed strain E. coli BT-6 decreased due to the knocked out of ptsG (Fig. 3B).The major byproduct of E. coli BT-5 was xylonate (12.30g/L) (Additional file 1: Figure S1), which can be transformed by lactose induced XylD and KdcA into 3,4-DHB and then reduced to 1,2,4-BT.Expression of xylD and kdcA is under control of P T7 in E. coli BT5 and BT-6 and lactose inducible.Besides xylose utilization, the catabolism of lactose in E. coli is also repressed by glucose due to carbon catabolite repression [7].Delayed response to induction often occurs during fermentation using lactose as inducer and glucose as carbon source.Although the consumption of xylose was not improved obviously, the inactivation of ptsG could enhance the utilization of lactose by E. coli BT-6 (Fig. 3B) and thus may also increase the expression of xylD and kdcA.As shown in Fig. 3B, 9.80 g/L 1,2,4-BT was obtained by E. coli BT-6 with a yield of 0.77 mol/mol xylose and accumulation of xylonate decreased to 4.14 g/L (Additional file 1: Figure S1).The enhanced production of 1,2,4-BT by E. coli BT-6 may due to the higher expression of xylD and kdcA by higher lactose utilization.The α-ketoacid decarboxylase KdcA used in this study was from L. lactis, whose optimum growth temperature is 30 ℃.The fermentation temperature for 1,2,4-BT production was tentatively adjusted to 30 ℃ and a slightly higher 1,2,4-BT concentration of 10.76 g/L was obtained with a yield of 0.86 mol/mol xylose from 17.67 g/L xylose (Fig. 3C).Thus, the 1,2,4-BT fermentation was conducted at 30 °C in subsequent experiments.

Integration of xylonate synthesis genes into E. coli genome to increase 1,2,4-butanetriol production
Expression of exogenous gene based on plasmid may increase the metabolic burden of recombinant E. coli [29].Expression of xylB and xylC based on plasmid pETP tac -xylBC may increase the metabolic burden of recombinant E. coli.As shown in Fig. 3B, the relief of carbon catabolite repression through deletion of ptsG enhanced lactose utilization and increased the production of 1,2,4-BT.It was reported that deletion of mgsA, the methylglyoxal synthase coding gene, can also effectively weaken the carbon catabolite repression [30].Thus, xylB and xylC under the control of P T7 were knocked into the position of ptsG and mgsA in E. coli 5KI genome for simultaneously relieving catabolite repression and expression of key enzymes for 1,2,4-BT production, resulting in E. coli BT-7 and E. coli BT-8, respectively.The endogenous xylose isomerase coding gene xylA is under the control of P xyl and can be induced by xylose in E. coli [31].The xylB and xylC were also directly knocked into the position of xylA and under the control of P xyl in E. coli 5KI, resulting in strain E. coli BT-9 (Fig. 4A).As shown in Fig. 4B, the strain E. coli BT-9 exhibited the best performance in 1,2,4-BT production.1,2,4-BT at a concentration of 12.85 g/L was obtained from 20.7 g/L xylose Fig. 6 1,2,4-BT production through fed-batch fermentation with E. coli BT-10 using xylose or CCH as the substrate.A Time-course of fed-batch fermentation by E. coli BT-10 in 7.5-L bioreactor with xylose as the substrate.The experiment was conducted in 7.5-L bioreactor with 5 L LB broth containing 30 g/L xylose, 10 g/L glucose and 10 g/L lactose at 400 rpm, 1.5 vvm and 30 °C.Xylose concentration was adjusted to 30 g/L when lower than 10 g/L.The experiment was conducted in triplicate.A representative time-course is reported herein.B Time-course of fed-batch fermentation by E. coli BT-10 in 7.5-L bioreactor with CCH as the substrate.The experiment was conducted in 7.5-L bioreactor containing 5 L LB broth with CCH (the final concentration of xylose was adjusted to 30 g/L) and whey powder (the final concentration of lactose was adjusted to 10 g/L) at 400 rpm, 1.5 vvm and 30 °C.Xylose concentration was adjusted to 30 g/L when lower than 10 g/L.The experiment was conducted in triplicate.A representative time-course is reported herein with a yield of 0.88 mol/mol xylose in 24 h (Additional file 1: Figure S2).

Fed-batch fermentation of E. coli BT-9 to produce 1,2,4-butanetriol in 1-L bioreactor
Then, fed-batch fermentation was carried out to produce 1,2,4-BT with E. coli BT-9.E. coli BT-9 was cultured in a 1-L bioreactor with 30 g/L xylose, 10 g/L lactose and 10 g/L glucose.Solid xylose was added under non-sterile condition when xylose concentration was lower than 10 g/L.After 48 h fed-batch fermentation, 1,2,4-BT at a concentration of 23.55 g/L was obtained from 46 g/L xylose with a yield of 0.72 mol/mol xylose.(Fig. 5A).

Knockout of pgi to strengthen NADPH supply and 1,2,4-butanetriol production
XylB catalyzes xylose into xylonolactone and generate NADH.However, YqhD required for 1,2,4-BT production is NADPH-dependent.Glucose can enter into EMP pathway to produce NADH or enter into pentose phosphate pathway to produce NADPH.Thus, the pgi encoding glucose phosphate isomerase was deleted in E. coli BT-9 to block the EMP pathway and enhance glucose metabolism via the HMP pathway to provide more NADPH required for 1,2,4-BT synthesis.The obtained E. coli strain BT-10 was used for 1,2,4-BT production in 1-L bioreactor.As shown in Fig. 5B, the 1,2,4-BT production of E. coli BT-10 increased to 27.2 g/L with a yield of 0.77 mol/mol xylose.Compared with E. coli BT-9, the accumulation of 3,4-DHB decreased 14.58% in E. coli strain BT-10 (Additional file 1: Figure S3).Then, the fed-batch fermentation of 1,2,4-BT by E. coli BT-10 was conducted in a 7.5-L bioreactor.Both the growth of E. coli BT-10 and the xylose consumption rate increased in the 7.5-L bioreactor, which may due to the improved dissolved oxygen during the enlargement of fermentation volume.As shown in Many biotechnological routes have been developed for the fermentative production of 1,2,4-BT.The synthesis of 1,2,4-BT from xylose or arabinose requires relatively few steps and results in low carbon loss, and thus has been intensively investigated in recent years [16][17][18][19][20][21][22]28,[32][33][34][35][36].A series of metabolic strategies such as screening enzymes with high activities, blocking the branch pathways, and enhancing the expression of the key enzymes, have been applied to improve 1,2,4-BT production from xylose or arabinose.CCH containing xylose, arabinose and glucose is an ideal substrate for 1,2,4-BT production.In this work, the encoding genes of XylB, XylC, XylD and KdcA were integrated into the genome of E. coli W3110 (DE3).Besides xylose, xylonolactone and xylonate, XylB, XylC and XylD from C. crescentus are also active on arabinose, arabinolactone and arabinonate, respectively [37].KdcA from L. lactis catalyzes the decarboxylation of both 2-keto-3-deoxy-xylonate and 2-keto-3-deoxy-arabinonate [38].Although the catabolic genes for arabinose utilization were not deleted in E. coli BT-10, these four heterologous enzymes and endogenous alcohol dehydrogenase YqhD may efficiently redirect the metabolic flux of arabinose from central metabolism to 1,2,4-BT production (Fig. 1).To eliminate catabolite repression and enhance the supply of NADPH for YqhD catalyzed 1,2,4-BT production with glucose, the ptsG and pgi genes were also deleted in E. coli W3110 (DE3).The final recombinant strain E. coli BT-10 can produce 43.4 g/L 1,2,4-BT from CCH with a productivity of 1.09 g/[L•h].Compared with other strains constructed for 1,2,4-BT production (Table 2), E. coli BT-10 has significant advantages of high 1,2,4-BT concentration, productivity, yield and efficient utilization of cheap substrate CCH.

Conclusion
In summary, a systematically metabolic engineered strain E. coli BT-10 was constructed to produce 1,2,4-BT.1,2,4-BT at a concentration of 36.63 g/L and a productivity 1.14 g/[L•h] was produced by E. coli BT-10 with xylose as the substrate.E. coli BT-10 can also use glucose in CCH for growth and transform xylose and arabinose into 1,2,4-BT.1,2,4-BT of 43.4 g/L was produced from CCH with a productivity of 1.09 g/[L•h].The process presented here is both a good example for efficient resource utilization of CCH and a promising alternative for industrial 1,2,4-BT production.

Fig. 2 Fig. 3
Fig. 2 Performance of different recombinant E. coli strains in 1,2,4-BT production from xylose.The experiment was carried out in the 300 mL shake flask containing 50 mL LB broth with 10 g/L xylose and 2 g/L lactose at 37 °C and 180 rpm for 24 h.Values are the average ± SD (n = 3 independent experiments)

Fig. 4 Fig. 5
Fig. 4 Selection of integration site of xylBC to increase 1,2,4-BT production.A Scheme of the integration mode of xylBC in E. coli BT-7, E. coli BT-8, and E. coli BT-9.B Performance of E. coli BT-7, E. coli BT-8, and E. coli BT-9 in transforming xylose into 1,2,4-BT.The fed-batch fermentation was carried out in 300 mL shake flask at 180 rpm and 30 °C.When xylose concentration was lower than 5 g/L, 10 g/L xylose was added.Values are the average ± SD (n = 3 independent experiments)

Table 1
Bacterial strains and plasmids used in this work a Cm r chloramphenicol resistant, Km r kanamycin resistant, Spe r spectinomycin resistant b CGSC Coli Genetic Stock Center, Yale university plasmid gene preservation center